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Abstract

The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish
heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such
samples as it records high-contrast images of entire planes within large samples at once. However,
in order to obtain images of different planes, it has been necessary to move the sample relative to
the fixed focal plane of the detection objective lens. This mechanical movement limits speed,
precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote
focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have
thereby achieved flexible volume imaging at much higher speeds than previously reported. Our
high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we
imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume
scans per second. Movements, shape changes and signals across the entire volume can be followed
which has been impossible with existing reconstruction techniques.

Figures (9)

Fig. 1Three different approaches to volume imaging in SPIM. (a) Volume image data in SPIM
can be recorded by scanning the object through the stationary light sheet and aligned focal plane of
the detection objective. Alternatively, the light sheet can be scanned through the sample by
synchronized adjustment of the focal plane, either by repositioning the detection lens (b) or remote
focusing (c), e.g. by a tunable lens to image the illuminated plane onto the camera.

Fig. 2A SPIM setup has been modified to include a scan mirror and an ETL lens: The light
sheet is generated by focusing a line into the back focal plane of the illumination lens using a
cylindrical lens. A motorized scan mirror displaces the light sheet along the detection optical
axis. The image plane is displaced by changing the focal length of the ETL in-between the two relay
lenses. Illumination light is shown in blue, fluorescence in green.

Fig. 3The ETL-SPIM setup exhibits a wide scan range and good image quality across almost the
whole field. (a) The ETL’s refractive power DETL
= 1/fETL as a function of the applied current (measurement by
Optotune AG). (b, c) The dependence of the position of the focal plane on currents
IETL for the 10×/0.3 lens (b) and the 20×/0.5 lens (c).
(d–f) A grating with a period of 100μm imaged with a 20×/0.5 lens for three
different currents: (d) IETL = 0mA, (e)
IETL = 160mA, and (f) IETL =
320mA. The red crosses indicate the lower/right edge of the line that were located to measure the
magnification and the distortion.

Fig. 4The dynamic behavior of the ETL in response to different driving signals. (a) The
image plane is scanned by applying a sawtooth current IETL. The maximum
intensity of the image is shown in red. Peaks can be observed whenever the image plane and the
light-sheet zLS coincide. (b) The maximum value taken of the images
p(t) plotted against the position of the light-sheet
zLS and the time point within each ETL-period
trel. The line at −90μm for
trel ≈ 0μm and 100μm is an artifact created by
an out-of-focus cluster of spheres. (c–f)
max(p(trel, zLS)) for
sawtooth signals IETL at νETL
= 20Hz (c), νETL = 40Hz (d), a lowpass-filtered
sawtooth at νETL = 40Hz (e) and a sine at
νETL = 40Hz (f).

Fig. 5Frequency-dependent amplitude for the 20× objective and phase of the
focal length of the ETL. The amplitude (a) and the phase shift (b) of the axial position of
the image plane. The ETL was driven with a sinusoidal signal. The crosses mark measurement results.
Exponentially decaying functions were fitted to the data.

Fig. 6Large volume scan with an ETL through the head of a zebrafish (
Media 1). (a)
Slowly varying ramp signals can be used for remote scanning through large volumes. Thin optical
sections are recorded by flashing the illumination only for a short time when the rolling shutter
exposes the full camera chip. (b,c) A stack of images of the vascular system in the brain of a
zebrafish imaged by remote scanning (blood cells in red, vasculature in green). The figure shows a
maximum projection along the detection axis overlaid with a bright-field image (b) and a
three-dimensional rendering (c) which is also shown in Media
1. The data was recorded with the 10× lens.

Fig. 7Dynamic calibration ensures perfect overlap of the light sheet and the image plane over the
full period for high-speed volume scans ( Media
2). (a) Maximum projection of the image stack across the
light sheet axis y over one period. Media
2 shows the full images. (b) Driving signals for the ETL,
IETL, and the piezo-mirror UPM. (c) Quality
parameter Q̄j that measures the overlap over one period for
different parameters uj and ϕj
grouped in five subsets depending on the offset U0,j
which is changed in steps dU. The maximum value is reached for
U0 = Uc, ϕ
= ϕc and u =
uc and marked by an arrow. The image stack shown in (a) was recorded
with these parameters. The data was recorded with the 20× lens.

Fig. 8Sinusoidal driving signals offer two possibilities to increase either spatial or temporal
sampling. The green plot indicates the position of the image plane along the optical axis
(vertical axis) over time (horizontal axis). The camera records images with constant temporal
spacing. The timepoints are marked by red dots and the number i of the frame within
the ETL period. m indicates the overall number of frames. In the two cases shown,
either: (a) Each plane is imaged twice during each period, e.g. i = 1 and
14, i = 2 and 13, ..., or (b) planes imaged in the second half are lying
between the planes imaged in the first half of each period.

Fig. 9Movies of multiple planes in a beating zebrafish heart can be acquired synchronously (
Media 3 &
Media 4). (a,b,c)
Three series of images taken in different planes. The temporal spacing between two frames taken in
the same plane is 1/fETL = 1/30s. The corresponding movie is
available as Media 3. The
relative positions of the planes along the detection axis z can be inferred from
Fig. 8(b) where the corresponding frames are marked by
*. Scalebar: 50 μm. (b) A three-dimensional reconstruction. Units are microns. The
corresponding movie is available as Media
4).